Identifying the causal agent of necrotizing hepatopancreatitis in shrimp: Multilocus sequence analysis approach

Abstract

The causal agent of the necrotizing hepatopancreatitis (NHP) disease in penaeid shrimp and other crustaceans was studied by sequencing its genome and locating particular biomarker genes that could contribute to a better taxonomic classification. Hepatopancreas of shrimp with clear signs of NHP was used as infective inoculum. The disease was reproduced and the causal agent was isolated by Percoll gradient. Genomic DNA was extracted, purified, submitted to high throughput sequencing and de novo assembled. Thereafter, strategies aimed to improve the taxonomic classification of the pathogen were used. A phylogenetic classification by multilocus sequence analysis (MLSA) was performed using the complete gene sequences of 16S rRNA, gyrase subunit alpha (gyrA), gyrase subunit beta (gyrB), RNA polymerase subunit beta (rpoB), ATP synthase subunit beta (atpD) and recombinase subunit alpha (recA). Phylogenetic analysis confirmed with a greater degree of confidence that the causal agent of NHP in shrimp belongs to the Holosporaceae family, with similarity to some species considered as pathogens of other arthropods.